Study Of The Effect And Mechanism Of Corilagin On The TLR2Signaling Pathway In HSV-1Induced Encephalitis

PART Ⅰ The change of toll-like receptors after HSV-1infected microgliaObjective:Study the expression of toll-like receptors after HSV-1infected microglia.Methods:BV2cells were infected by HSV-1, then Real-time PCR was used to detect the expression of TLR2/TLR3/TLR9mRNA.Results:HSV-1infected group showed increase in expression of TLR2/TLR3/TLR9mRNA, the difference were significant compare with that of in normal group (P<0.01).Conclusions:TLR2, TLR3and TLR9were involved in the recognition of Herpes simplex virus-1in BV2cells. PART Ⅱ The change of toll-like receptor2and downstream mediators after HSV-1infected microgliaObjective:Study the expression of toll-like receptor2and downstream mediators after HSV-1infected microglia. Methods:BV2cells were infected by HSV-1and Malp2, then Flow cytometry (FCM) was used to measure the levels of TLR2and Western blot was used to detect the levels of TIRAP、MyD88、TRAF6、P38、NEMO and NF-κ Bp65,Real-time PCR was used to detect the mRNA expression of TLR2、TIRAP、 MyD88、TRAF6、P38and NEMO. ELISA was also used to measure the levels of IL-6and TNF-α. Results:1、TLR2and its downstream mediators were significantly increased after Malp2stimulated BV2cells, the difference were significant compare with that of in normal group (P<0.05).2、TLR2and its downstream mediators were significantly increased after HSV-1infected BV2cells, the difference were significant compare with that of in normal group (P<0.05). Conclusions:TLR2and its downstream mediators initiated innate responses to herpes simplex virus type1in BV2cells. Keywords:herpes simplex virus type1, microglia, toll-like receptor2signaling pathway PART Ⅲ The effect of Corilagin on toll-like receptor2signaling pathway after HSV-1infected microgliaExperiment1The effect of Corilagin on toll-like receptor2signaling pathway after HSV-1infected normal microgliaObjective:Study the effect of Corilagin on toll-like receptor2signaling pathway after HSV-1infected microglia. Methods:BV2cells were infected by HSV-1and Malp2, then cells treated by Corilagin for24h. Flow cytometry (FCM) was used to measure the levels of TLR2and Western blot was used to detect the levels of TIRAP、 MyD88、TRAF6、P38、NEMO and NF-κ Bp65,Real-time PCR was used to detect the mRNA expression of TLR2、TIRAP、MyD88、TRAF6、P38and NEMO. ELISA was also used to measure the levels of IL-6and TNF-a. Results:1、After Corilagin treated BV2cells that stimulated by Malp2,TLR2and its downstream mediators were significantly decreased, the difference were significant compare with that of in normal saline group (P<0.05).2、After Corilagin treated BV2cells that infected by HSV-1,TLR2and its downstream mediators were significantly decreased, the difference were significant compare with that of in normal saline group (P<0.05)Conclusions:Corilagin can inhibit the activation of TLR2and its downstream mediators after HSV-1infected BV2cells. Experiment2The mechanism of Corilagin on toll-like receptor2signaling pathway after HSV-1infected microgliaObjective:Study the mechanism of Corilagin on toll-like receptor2signaling pathway after HSV-1infected microglia. Methods:When TLR2was excessive or TLR2was silent, BV2cells were infected by HSV-land Malp2, then treated by Corilagin for24h. Western blot、Real-time PCR and ELISA were used to measure the levels of TLR2downstream mediators. Results:1、When TLR2was excessive,after Corilagin treated BV2cells that stimulated by Malp2,TLR2downstream mediators were decreased, the difference were significant compare with that of in normal saline group (P<0.05).2、When TLR2was silent, after Corilagin treated BV2cells that infected by HSV-1,the levels of MyD88and TRAF6were increased compare with that of in normal saline group (P<0.05), but the difference of TRIAP was not significant (P>0.05).however, the levels of P38、NEMO、NF-κBp65、IL-6and TNF-α were decreased, the difference were significant compare with that of in normal saline group (P<0.05). Conclusions:Corilagin might inhibit NEMO, P38, p-P38, NF-κB, TNF-α, IL-6directly or via other TLRs and the inhibition of Corilagin to the TIRAP/MyD88-TRAF6pathway needed TLR2. PART IV The effect of Corilagin on toll-like receptor2signaling pathway after HSV-1infected murine brainObjective:Study the effect of Corilagin on toll-like receptor2signaling pathway after HSV-1infected murine brain. Methods:Murine model of HSV-1was set up following injection of HSV-1samples in Balb/c mice brains. These subjects were randomly divided in the following groups:normal group, Corilagin group, Malp2+normal saline group, Malp2+Corilagin group, HSV-1+normal saline group, HSV-1+Corilagin group. HE stain was used to study brain samples, immunohistochemical staining was used to detect the expression of TLR2, Western blot was used to detect the levels of TIRAP、MyD88、TRAF6、P38、NEMO and NF-κ Bp65,Real-time PCR was used to detect the mRNA expression of TLR2、TIAP、 MyD88、TRAF6、P38and NEMO,ELISA was also used to measure the levels of IL-6and TNF-α. Results:1、pathological changes in the brain tissues were minimum in Corilagin-treated group.2、Malp2+normal saline group and HSV-1+normal saline group compare with that of in normal group,TLR2and its downstream mediators were significant increased (P<0.05).3%Malp2+Corilagin group compare with Malp2+normal saline group, TLR2and its downstream mediators were significant decreased (P<0.05).4、HSV-1+Corilagin group compare with HSV-1+normal saline group, TLR2and its downstream mediators were significant decreased (P<0.05)Conclusions:Corilagin can effectively prevent HSV-1induced brain damage through TLR2signaling pathway.